Method of purifying a hydrophobin present in a hydrophobin-containing solution

ABSTRACT

The invention relates to a method of purifying a hydrophobin present in a hydrophobin-containing solution. According to the invention the solution is contacted with a surface for the adsorption to that surface, and separated from a solution depleted in hydrophobin. Subsequently the surface is contacted with a solution containing a surfactant at a temperature lower than 90° C. Desorbed hydrophobin is separated from said surface.

This application is a national stage application of PCT/NL01/00083,filed on Feb. 2, 2001, which claims foreign priority from UK 0002661.7,filed on Feb. 4, 2000.

The present invention relates to a method of purifying a hydrophobinpresent in a hydrophobin-containing solution.

Hydrophobins are proteins known for their capability of forming awater-insoluble coating on a surface of an object. The adherence is sostrong that the coating can not be removed by boiling in a 2% sodiumdodecylsulfate (SDS) solution. Indeed, it has been suggested to coat asurface of, for example a biosensor, with a hydrophobin to modify thehydrophobic/hydrophillic nature of said surface. Ahydrophobin-containing solution should be handled with care, as actionssuch as shaking result in turbid solutions containing hydrophobinaggregates which affect a uniform coating of a surface. For theapplication of a hydrophobin on a significant scale, an industrial scalemethod is necessary for purifying a hydrophobin present in ahydrophobin-containing solution, such as growth medium of afermentation. The method according to the state of the art relies on theuse of trifluoroacetic acid (TFA), which is for environmental and safetyreasons not desirable.

To this end the present invention provides a method according to thepreamble, characterized in that the hydrophobin-containing solution iscontacted with a surface for adsorption to that surface, separation ofthe surface carrying adsorbed hydrophobin and a solution depleted inhydrophobin, after which the surface carrying adsorbed hydrophobin iscontacted with a solution containing a surfactant at a temperature lowerthan 90° C. to solubilize the hydrophobin adsorbed to said surface andseparating a hydrophobin-enriched surfactant-comprising solution fromsaid surface.

Surprisingly it has been found that, provided that the temperaturecondition is met, hydrophobin can be eluted from a surface to which itis adsorbed. It goes without saying that the surface is the surface ofan object having a high surface to volume ratio. To prevent anyirreversible changes in structure, rendering the hydrophobin insoluble,the surface carrying hydrophobin should not be subjected to temperaturesexceeding 90° C. before being contacted with the solution containingsurfactant.

Hydrophobins are a well-defined class of proteins (ref. 1) capable ofself-assembly at a hydrophobic-hydrophilic interface, and having aconserved sequenceX_(n)—C—X₅₋₉—C—C—X₁₁₋₃₉—C—X₈₋₂₃—C—X₅₋₉—C—C—X₆₋₁₈—C—X_(m)X, of course, represents any amino acid, and n and m, of course,independently represent an integer. In general, a hydrophobin has alength of up to 125 amino acids. The cysteine residues (C) in theconserved sequence are part of disulfide bridges. In the presentinvention, the term hydrophobin has a wider meaning to includefunctionally equivalent proteins, and encompasses a group of proteinscomprising the sequence or parts thereofX_(n)—C—X₁₋₅₀—C—X₀₋₅—C—X₁₋₁₀₀—C—X₁₋₁₀₀—C—X₁₋₅₀—C—X₀₋₅—C—X₁₋₅₀—C—X_(m)still displaying the characteristic of self-assembly at ahydrophobic-hydrophilic interface resulting in a protein film. Inaccordance with the definition of the present invention, self-assemblycan be detected by adsorbing the protein to Teflon and use CircularDichroism to establish the presence of a secondary structure (in generalα-helix) (ref. 2). The formation of a film can easily be established byincubating a Teflon sheet in the protein solution followed by at leastthree washes with water or buffer (ref. 3). The protein film can bevisualised by any method, such as labeling with a fluorescent compoundor by the use of fluorescent antibodies, as is well established in theart. m and n may have values ranging from 0 to 2000. Included in thedefinition are fusion-proteins of a hydrophobin and another protein assuch recombinant proteins may similarly be purified with the methodaccording the present invention.

WO-A-9641882 describes in a general fashion purification of hydrophobinsusing precipitation and chromatographic methods, in particularprecipitation using methanol, ethanol, acetone and ammonium sulphate,and ion exchange and hydroxy apatite chromatography. SDS is mentioned ashelpful when isolating hydrophobin from the cells in which it isproduced and from culture medium in which they are present.

Martin G. G. et al. describe in J. Am. Chem. Soc. vol. 39 (1), p.347-348 (1998) a method of purifying hydrophobin using preparativeelectrophoresis, followed by hydroxy apatite chromatography to removeSDS.

Preferably the temperature is lower than 60° C., such as lower than 40°C., in particular lower than 20° C., and more preferably lower than 10°C.

In general, is the concentration of the surfactant between 0.001% and 5%(w/v), advantageously between 0.01% and 1.0% (w/v), preferably between0.02% and 0.1%.

Suitable concentrations surfactant can easily be determined usingvarious spectroscopic techniques such as circular dichroism (CD) such asdescribed by De Vocht et al (ref. 2) or Infra Red (IR) spectroscopy(ibid). Alternatively, it is possible to determine a suitableconcentration surfactant by simple trial and error: A surface is coatedwith a hydrophobin-containing solution, and said surface is treated withthe solution containing the surfactant after which the presence ofhydrophobin is investigated using, for example, fluorescence-based orradioactivity-based techniques. Although it is possible to use suitablylabelled antibodies against hydrophobin, it is easier to coat thesurface with labelled hydrophobin and detect the amount of labelremained in comparison with a surface treated with the same solution butwithout surfactant.

According to a further embodiment, the hydrophobin is solubilized undera pressure of at least 1.1 Bar.

Elevated pressures facilitate the elution of the adsorbed hydrophobinfrom the surface carrying adsorbed hydrophobin.

According to another preferred embodiment, the pressure is reducedduring adsorption of the hydrophobin at the surface.

Reducing the pressure during adsorption facilitates the adsorption ofthe hydrophobin to the surface. In general, the pressure of thehydrophobin-containing solution will have a pressure of at least 1.1 Barwhen the solution is first contacted with the surface.

In principle, the surface at which the hydrophobin is to be adsorbed,may be of any material, such as glass or plastic. Preferably the surfacehas a contact angle for water larger than 60°. Such a contact anglemakes the surface very suitable for adsorbing hydrophobin present in thehydrophobin-containing solution.

According to a preferred embodiment, the surface is the surface of anobject having a high surface-to-volume ratio.

This allows for the purification of hydrophobin in a relatively smallvolume.

The invention will now be illustrated by way of the following examples.

Methods

A) Secondary Structure Measurements

-   -   The secondary structure of a hydrophobin was studied with        circular dichroism spectroscopy (CD). The CD-spectra were        recorded over the wavelength region 190-250 nm on an Aviv 62A DS        CD spectrometer (Aviv Associates, Lakewood, N.J., USA), using a        1-mm quartz cuvette and following a known procedure (2). The        sample compartment was continuously flushed with N₂ gas and the        temperature was kept constant at 25° C. 10 scans were averaged,        using a bandwidth of 1 nm, a stepwidth of 1 nm, and 1 sec        averaging per point. The spectra were corrected using a        reference solution without the protein. Typically a protein        concentration of 10 μM in 50 mM phosphate pH 7.0 was used. For        spectra of SC3 bound to a hydrophobic support, 130 nm        unstabilized colloidal Teflon spheres (Dupont de Nemours,        Geneva, Switzerland) in water were added to the solution.        B) Binding to Teflon    -   The coating of Teflon (Norton Fluorplast B. V., Raamsdonksveer,        The Netherlands) by SC3 was assessed essentially as described by        Wösten et al. (3). Thoroughly cleaned (ref. 3) Teflon sheets        were incubated for 16 hours in 20 μg/ml ³⁵S-labelled hydrophobin        in water, followed by three washes with water for 10 minutes        each. The amount of adsorbed ³⁵S-labelled protein were        determined by scintillation counting.

EXAMPLE 1

50 μg/ml SC3 in 50 mM phosphate buffer (pH=7.0) was mixed with 130 nmunstabilized colloidal Teflon spheres (Dupont de Nemours, Geneva,Switzerland) at 25° C. SC3 adsorbed to the surface of the Teflon andattained the α-helical state (calculated surface coverage 9%).

The spheres were treated with 0.1% w/v Tween-20 or 0.1% v/w Tween-80 at25° C. for 10 minutes and spun down (1 min; 10,000 g). Substantially100% of SC3 desorbed after addition of the detergent and attained themonomeric state. As expected, in the absence of detergent SC3 remainedadsorbed (in the α-helical state as determined by CD).

EXAMPLE 2

Teflon sheets (2 cm², thickness 0.25 mm) were incubated in 20 μg/ml³⁵S-labelled SC3 overnight at room temperature. The SC3-coated sheetswere subsequently washed with water at room temperature. The sheets werethen treated with 2% Tween 20 (pH 7.0) or water (control), either atroom temperature or 100° C. (control) for 30 min. After this treatment,the sheets were removed. The amount of radioactive SC3 released into thesupernatant obtained after centrifugation (1 min; 10,000 g) wasdetermined. Percentages are relative to the amount of radioactivityoriginally bound to the sheet.

TABLE 1 The amount of radioactive SC3 released into the supernatantobtained after centrifugation. % SC3 released room temperature 100° C.2% Tween 20 78% 6% Water (control) 6% 7%

This shows that a surfactant may be used to elute a hydrophobin providedthe temperature requirement is met.

References

-   1. Wessels, J. G. H. (1997) in Adv. Microb. Physiol. 38, 1-45.-   2. De Vocht, M. L., et al. (1998) in Biophys. J. 74, 2059-68.-   3. Wösten, H. A. B., et al. (1994) in Embo. J. 13, 5848-54.

1. A method for purifying hydrophobin, said method comprising: a)contacting the hydrophobin-containing solution with surface foradsorption to that surface, b) separating the surface carrying adsorbedhydrophobin and solution depleted in hydrophobin, c) contacting thesurface carrying adsorbed hydrophobin with a solution containing asurfactant at a temperature lower than 90° Centigrade to solubilize thehydrophobin adsorbed to said surface, and d) separating ahydrophobin-enriched surfactant-comprising solution from said surface.2. Method according to claim 1, wherein the temperature is lower than60° C.
 3. Method according to claim 1, wherein the temperature is lowerthan 40° C.
 4. Method according to claim 1, wherein the temperature islower than 20° C.
 5. Method according to claim 1, wherein thetemperature is lower than 10° C.
 6. Method according to claim 1, whereinthe concentration of the surfactant is between 0.001% and 5% (w/v). 7.Method according to claim 1, wherein the concentration of the surfactantis between 0.01% and 1.0% (w/v).
 8. Method according to claim 1, whereinthe concentration of the surfactant is between 0.02% and 0.1% (w/v). 9.Method according to claim 1, wherein the hydrophobin is solubilizedunder a pressure of at least 1.1 Bar.
 10. Method according to claim 1,wherein during adsorption of the hydrophobin at the surface, thepressure is reduced.
 11. Method according to claim 1, wherein thesurface has a contact angle for water larger that 60°.